LPXD1 (At4g05210) is one of two Arabidopsis paralogs of the bacterial UDP-3-O-acylglucosamine N-acyltransferase LpxD, the third enzyme of the lipid A precursor pathway, which adds an (R)-3-hydroxyacyl chain from acyl carrier protein to the glucosamine amine of UDP-3-O-acyl-GlcN. It is a nuclear-encoded hexapeptide-repeat (left-handed beta-helix) acyltransferase with an N-terminal mitochondrial transit peptide, and an N-terminal GFP fusion is targeted to mitochondria. The catalytic histidine is conserved, but an arginine implicated in acyl-ACP binding is replaced by lysine and two aromatic acceptor-binding residues are missing. Unlike its paralog LPXD2, loss of LPXD1 does not lower the pathway intermediate lipid X (2,3-diacylglucosamine 1-phosphate), and null plants have no visible phenotype. LPXD1 is therefore at most a minor contributor to lipid X synthesis, and whether it has LpxD activity, a different acyltransferase activity (such as LpxA-like UDP-GlcNAc acylation) or no catalytic role is unknown.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | MODIFY | Summary: Cytoplasm, from an ARBA machine-learning rule. Reason: True in the GO sense (mitochondria are part of the cytoplasm) but less specific than the evidence supports. A 5'-terminal LPXD1-GFP fusion shows a mitochondrial pattern and the protein has a predicted N-terminal mitochondrial transit peptide, so the informative term is mitochondrion (GO:0005739), which the gene already carries from IDA, IBA and IEA rows. Proposed replacements: mitochondrion Supporting Evidence: PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Mitochondrion propagated by PAINT from the plant LpxD node PTN008106513, whose experimental grounding is the GFP localisation of both Arabidopsis LpxD paralogs. Reason: The node is a plant-specific mitochondrial LpxD clade. The target appearing among its own sources reflects its IDA localisation, which is expected and not circular. Consistent with the GFP data and the predicted transit peptide. Supporting Evidence: PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC file:ARATH/LPXD1/LPXD1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:21709257} |
| GO:0005739 mitochondrion | IDA PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | ACCEPT | Summary: Mitochondrion from a 5'-terminal LPXD1-GFP fusion imaged with MitoTracker in root cells of transgenic Arabidopsis (Li et al. 2011). Reason: Direct experimental localisation. The full-length fusion was too dim to image, so the result rests on the N-terminal region fused to GFP; this shows that the N-terminal presequence is a functional mitochondrial targeting signal, which is the expected determinant of the protein's location. The lipid X pool that this pathway makes is also enriched in mitochondria. Mitochondrion is the right level of precision; no sub-mitochondrial location has been tested. Supporting Evidence: PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC PMID:21709257 Full-length GFP fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK did not emit sufficient GFP fluorescence to permit localization. PMID:21709257 Lipid X levels in mitochondria were 3- and 48-fold higher than in chloroplasts or whole cell homogenates, respectively. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrion from the UniProt subcellular location mapping; the UniProt line is backed by Li et al. 2011 (ECO:0000269). Reason: Same claim as the IDA row; supported by GFP localisation. Supporting Evidence: file:ARATH/LPXD1/LPXD1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:21709257} PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC |
| GO:0009245 lipid A biosynthetic process | IEA GO_REF:0000002 | UNDECIDED | Summary: Lipid A biosynthetic process from the InterPro2GO mapping of IPR007691 (LpxD family). This term on plant LpxD proteins was flagged by Wei et al. 2020 as a probable misannotation. Reason: The pathway itself is real in Arabidopsis (the RAR flag is refuted at the pathway level, and for the paralog LPXD2), but there is no evidence that LPXD1 does any of the work. In atlpxd1-1 null plants lipid X is not reduced, and adding atlpxd1-1 to atlpxb-1 does not lower the elevated lipid X. Residual lipid X in atlpxd2 atlpxb plants leaves open a minor LPXD1 contribution, and the authors also raise the possibility that LPXD1 is an LpxA-like enzyme, which would still place it in this pathway but at a different step. LPXD1 also carries substitutions at substrate-binding residues. Family membership alone does not settle it for a paralog that has diverged at these positions, so the claim can be neither accepted nor removed on current evidence. Supporting Evidence: PMID:21709257 The lipid X level in the atlpxd1-1 mutant was not reduced from that of wild type PMID:21709257 Introducing the atlpxd1-1 mutation into the atlpxb-1 mutant background did not lower its elevated lipid X levels PMID:21709257 The presence of some residual lipid X in this setting leaves open the possibility that AtLpxD1 generates a small portion of the lipid X pool. PMID:21709257 At present, we cannot exclude the intriguing possibility that AtLpxD1 has LpxA activity PMID:21709257 our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | REMOVE | Summary: Chloroplast predicted by AtSubP sequence analysis (TAIR). Reason: Contradicted by experiment. In mesophyll cells the Lpx-GFP fusions formed punctate signals that did not colocalise with chloroplasts, and in roots the LPXD1 fusion showed the mitochondrial pattern. The N-terminal presequence is a predicted mitochondrial transit peptide. The only chloroplast link is that some lipid X (in atlpxb-1) is found in chloroplasts, which concerns the lipid, not this enzyme. Supporting Evidence: PMID:21709257 In mesophyll cells, GFP signals were shown as 1 ΞΌm dots, but were not colocalized with chloroplasts PMID:21709257 However, 5β²-terminal fusions of AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK to GFP showed the same mitochondrial pattern as full-length AtLpxA and AtLpxC |
| GO:0016740 transferase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Transferase activity from InterPro2GO mapping of IPR018357 (hexapeptide transferase conserved site). Reason: True but uninformative. The more specific family-level activity, acyltransferase activity transferring groups other than amino-acyl groups (GO:0016747), is accepted in this review and entails it. Supporting Evidence: file:ARATH/LPXD1/LPXD1-uniprot.txt Belongs to the transferase hexapeptide repeat family. LpxD subfamily. |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | IEA GO_REF:0000002 | ACCEPT | Summary: Acyltransferase activity (non-aminoacyl) from InterPro2GO mapping of IPR007691 (LpxD). Reason: This is the most specific activity the evidence supports for LPXD1. It is a full-length hexapeptide-repeat acyltransferase of the LpxD subfamily, about 75% identical to LPXD2, with the catalytic histidine (E. coli His239 equivalent) conserved. Whether its acyl acceptor is UDP-3-O-acyl-GlcN (LpxD activity) or UDP-GlcNAc (LpxA-like activity, as Li et al. suggest) is unresolved, so the broad acyltransferase term is as precise as is warranted and should not be narrowed. Supporting Evidence: file:ARATH/LPXD1/LPXD1-uniprot.txt Belongs to the transferase hexapeptide repeat family. LpxD subfamily. file:ARATH/LPXD1/LPXD1-bioinformatics/RESULTS.md EcLpxD catalytic H239 -> AtLpxD1 H217, AtLpxD2 H216 (both conserved; these match the UniProt ACT_SITE positions 217 and 216). PMID:21709257 these proteins share 41 and 34% identity with E. coli LpxD ( Table 1 ), with conservation of key catalytic residues PMID:21709257 At present, we cannot exclude the intriguing possibility that AtLpxD1 has LpxA activity |
| GO:0103118 UDP-3-O-[(3R)-3-hydroxyacyl]-glucosamine N-acyltransferase activity | IBA GO_REF:0000033 | UNDECIDED | Summary: LpxD activity propagated by PAINT from node PTN000501148, seeded by E. coli LpxD (P21645) and the two Arabidopsis LpxD paralogs. Reason: The node placement is sound for the family, and LPXD1 lies inside the clade. However, there is target-specific evidence of divergence. The R293 residue implicated in acyl-ACP binding is lysine in LPXD1 (confirmed on the current gene model), two aromatic acceptor-binding residues are missing, and loss of LPXD1 does not change lipid X levels. The authors conclude that LPXD2 is the primary LpxD and leave open both a minor LpxD role and an LpxA-like activity for LPXD1. No in vitro assay or E. coli complementation of LPXD1 exists. The inherited activity is plausible but neither shown nor excluded, so the row is left undecided rather than accepted or removed. Propagation Review Root cause: UNRESOLVED Sources checked: PANTHER:PTN000501148 Β· LpxD ancestral node UNRESOLVED Node is correct for the family; LPXD1 is inside it but shows substitutions at acyl-ACP and acceptor binding residues and no mutant effect on lipid X, so inheritance of the exact activity is unresolved. UniProtKB:P21645 Β· E. coli LpxD SUPPORTS SOURCE BUT NOT TARGET Biochemically and structurally characterised LpxD; the donor annotation is sound, but whether it applies to this diverged paralog is untested. Supporting Evidence: PMID:21709257 R293 is conserved between E. coli LpxD and AtLpxD2, but is replaced by lysine in AtLpxD1. PMID:21709257 AtLpxD1 also lacks two aromatic residues in its N-terminal domain that are required for acceptor substrate binding file:ARATH/LPXD1/LPXD1-bioinformatics/RESULTS.md EcLpxD R293 -> AtLpxD1 K271, AtLpxD2 R270. PMID:21709257 The lipid X level in the atlpxd1-1 mutant was not reduced from that of wild type PMID:21709257 The presence of some residual lipid X in this setting leaves open the possibility that AtLpxD1 generates a small portion of the lipid X pool. |
| GO:0103118 UDP-3-O-[(3R)-3-hydroxyacyl]-glucosamine N-acyltransferase activity | IEA GO_REF:0000120 | UNDECIDED | Summary: LpxD activity from combined Rhea/EC mapping of the UniProt catalytic activity line. Reason: Same claim as the IBA row. The UniProt catalytic activity on this entry carries no experimental evidence (the protein name is "Probable"), and the reasons for doubt given on the IBA row apply. Supporting Evidence: PMID:21709257 R293 is conserved between E. coli LpxD and AtLpxD2, but is replaced by lysine in AtLpxD1. PMID:21709257 The lipid X level in the atlpxd1-1 mutant was not reduced from that of wild type |
| GO:0103118 UDP-3-O-[(3R)-3-hydroxyacyl]-glucosamine N-acyltransferase activity | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | UNDECIDED | Summary: LpxD activity by mutant phenotype, citing Li et al. 2011. Reason: The cited paper's mutant data do not show this activity for LPXD1. Lipid X, the downstream intermediate, is not reduced in the atlpxd1-1 null mutant, and atlpxd1-1 does not lower lipid X in the atlpxb-1 background; the paper attributes the activity to LPXD2. The only LPXD1-related signal is residual lipid X in atlpxd2 atlpxb plants, which the authors present as an open possibility. The full text was read, so this is not deferring to unseen evidence; but the activity is not excluded either, so the row is left undecided, consistent with the IBA and IEA rows for the same claim. Supporting Evidence: PMID:21709257 The lipid X level in the atlpxd1-1 mutant was not reduced from that of wild type PMID:21709257 Introducing the atlpxd1-1 mutation into the atlpxb-1 mutant background did not lower its elevated lipid X levels PMID:21709257 The presence of some residual lipid X in this setting leaves open the possibility that AtLpxD1 generates a small portion of the lipid X pool. PMID:21709257 These results suggest that AtLpxD2 is the primary UDP-3- O -acyl-GlcN N -acyltransferase in Arabidopsis |
| GO:2001289 obsolete lipid X metabolic process | IBA GO_REF:0000033 | UNDECIDED | Summary: Obsolete term "lipid X metabolic process" propagated from the plant LpxD node PTN008106513. Reason: QuickGO marks GO:2001289 obsolete ("made in error and is an unnecessary grouping term") with no replacement; the current equivalent claim is lipid A biosynthetic process (GO:0009245). For LPXD1 that claim is unresolved (see the GO:0009245 row) because loss of LPXD1 does not change lipid X, so no replacement is proposed and the row is left undecided. Propagation Review Root cause: UNRESOLVED Sources checked: PANTHER:PTN008106513 Β· plant LpxD node SOURCE STALE OR MISSING The propagated term is obsolete; the node's experimental grounding for lipid X metabolism comes from the LPXD2 mutant, and the LPXD1 mutant shows no lipid X change. Supporting Evidence: PMID:21709257 The lipid X level in the atlpxd1-1 mutant was not reduced from that of wild type PMID:21709257 The presence of some residual lipid X in this setting leaves open the possibility that AtLpxD1 generates a small portion of the lipid X pool. |
| GO:2001289 obsolete lipid X metabolic process | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | UNDECIDED | Summary: Obsolete term "lipid X metabolic process" by mutant phenotype, citing Li et al. 2011. Reason: The term is obsolete without replacement, and the single-mutant data do not show a phenotype for LPXD1. Lipid X is not reduced in atlpxd1-1 plants, and introducing atlpxd1-1 into atlpxb-1 does not lower the accumulated lipid X. However, the authors note that residual lipid X in the atlpxd2-1 atlpxb-1 background leaves open a minor contribution from AtLpxD1, so the experiment neither establishes nor excludes a role. Left undecided, consistent with the LpxD activity rows for this paralog. The UniProt disruption-phenotype line for this entry does not match the paper; see source_defects. Supporting Evidence: PMID:21709257 The lipid X level in the atlpxd1-1 mutant was not reduced from that of wild type PMID:21709257 Introducing the atlpxd1-1 mutation into the atlpxb-1 mutant background did not lower its elevated lipid X levels PMID:21709257 The presence of some residual lipid X in this setting leaves open the possibility that AtLpxD1 generates a small portion of the lipid X pool. |
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Download this section (compressed HTML)Q: Does recombinant LPXD1 acylate UDP-3-O-acyl-GlcN (LpxD activity), UDP-GlcNAc (LpxA-like activity) or neither, and does the R293K substitution change its acyl-ACP chain-length preference?
Q: Is LPXD1 expressed at a level, tissue or condition where it could contribute to lipid X synthesis, given that LPXD2 provides nearly all of it in seedlings?
Q: Is the 2011 claim that only LPXD2 has the N-terminal nucleotide-binding domain an artefact of the gene model used at the time?
Experiment: Express mature LPXD1 (without transit peptide) and assay with R-3-hydroxymyristoyl-ACP and either UDP-3-O-acyl-GlcN or UDP-GlcNAc; test complementation of E. coli lpxD and lpxA conditional mutants alongside LPXD2.
Hypothesis: LPXD1 has weak LpxD activity or LpxA-like activity.
Type: in vitro enzyme assay and bacterial complementation
Experiment: Generate atlpxd1 atlpxd2 and atlpxa atlpxd1 double mutants in a matched background and quantify lipid X by LC-MRM.
Hypothesis: LPXD1 accounts for the residual lipid X in atlpxa or atlpxd2 mutants.
Type: double mutant lipid profiling
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